The protein composition of synapses is exquisitely regulated to maintain healthy brain function. The synapse contains diverse set of centrally important transmembrane proteins, including neurotransmitter receptors, cell adhesion molecules, and ion channels. The precise architectural organization of these components establish postsynaptic function. The broad goal of this proposal is to understand how these critical postsynaptic proteins concentrate within excitatory synapses, and how regulation of their number and position contribute to mechanisms of neural plasticity in health and disease. Of particular importance is regulation of the AMPA-type glutamate receptors (AMPARs), because the number of activated AMPARs is controlled and modulated during many forms of neural plasticity. AMPARs diffuse freely on the neuronal surface membrane, and enter and exit the postsynaptic density (PSD) via this mechanism. In order to sustain synaptic strength, the synapse slows mobility of receptors to retain them. Unfortunately, despite intensive investigation, the mechanisms governing intrasynaptic receptor mobility remain unclear. Compelling data including high resolution imaging of receptor lateral movement suggest that binding to partners within the PSD, notably the scaffold PSD-95, is essential for receptor accumulation. However, numerical modeling and indirect experimental evidence suggest an alternative possibility, i.e. the synapse is so dense with proteins that this obstacle field prevens receptors from escaping. Nevertheless, this mechanism (intrasynaptic steric hindrance of receptors) is poorly understood and has not been systematically addressed. Motivated by these observations, I hypothesize that steric hindrance and biochemical binding in the PSD act in combination to regulate the mobility of synaptic transmembrane proteins like receptors. I have combined two single-molecule imaging approaches that permit discrimination of protein mobility within and around synapses with the necessary, nanometer-scale resolution. Using these approaches to monitor motion of a uniquely designed set of transmembrane protein probes will allow me to examine the influence of binding and steric hindrance separately. If the synaptic environment exerts steric influence on proteins entering the PSD, the effect should depend on protein size. The first set of experiments tests this prediction by altering the extracellular sizeof an otherwise identical binding-deficient transmembrane protein, and tracking their movements in the living synapse. I then assess whether steric hindrance in the extracellular and intracellular environment in the synapse could have different effects on receptor mobility, by altering the bulk size of the same protein on either side of the membrane and tracking their movements in the living synapse. The next experiments test the sufficiency of synaptic binding to slow transmembrane protein by following the movements of a small, generic transmembrane protein carrying a ligand which can bind to PSD- 95. These results greatly clarify important mechanisms controlling key proteins of the synapse.